Yamaguchi Tomoko, Naruishi Koji, Arai Hideo, Nishimura Fusanori, Takashiba Shogo
Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
J Cell Physiol. 2008 Nov;217(2):423-32. doi: 10.1002/jcp.21517.
Interleukin (IL)-6 has an important role in inflammatory diseases. Lysosomal enzymes cathepsins are widely expressed as cysteine proteases regulating inflammatory process. Caveolin-1 (Cav-1) is a scaffolding/regulatory membrane protein that interacts with signaling molecules. In this study, we investigated the role of Cav-1 on (1) the productivity, and (2) the enzymatic activity of cathepsin B and L in human gingival fibroblasts (HGFs) treated with IL-6 in the presence of soluble form of IL-6 receptor (sIL-6R). At first, we established the siRNA-mediated Cav-1 down-regulating in vitro systems by transient transfection of Cav-1 siRNA. The siRNA-mediated Cav-1 down-regulated cells were treated with IL-6/sIL-6R for indicated times. Then, cell lysates were collected, and examined the IL-6-induced signaling pathway, cathepsin B and L production, and measurement of cathepsins activity. To investigate the cathepsin L activity, cathepsin-(B + L) activity was measured after pretreatment with CA-074Me, a specific inhibitor for cathepsin B. We found that IL-6/sIL-6R enhanced significantly both production and activity of cathepsin B and L in HGFs. Interestingly, IL-6-mediated phosphorylation of both p44/42 MAPK and JNK was dramatically suppressed in Cav-1 down-regulated HGFs treated with IL-6/sIL-6R. In addition, both production and activity of cathepsin B and L were also significantly suppressed. Importantly, we demonstrated that JNK inhibition, but not p44/42 MAPK inhibition, significantly diminished IL-6/sIL-6R-induced cathepsin B and L production. Taken together, we concluded that IL-6/sIL-6R enhances cathepsin B and L production via IL-6/sIL-6R-mediated Cav-1-JNK-AP-1 pathway in HGFs. Our findings indicate that Cav-1 might be a therapeutic target for IL-6-mediated tissue degradation in periodontitis.
白细胞介素（IL）-6在炎症性疾病中发挥着重要作用。溶酶体酶组织蛋白酶作为调节炎症过程的半胱氨酸蛋白酶广泛表达。小窝蛋白-1（Cav-1）是一种与信号分子相互作用的支架/调节膜蛋白。在本研究中，我们研究了Cav-1在（1）人牙龈成纤维细胞（HGFs）中，在可溶性白细胞介素-6受体（sIL-6R）存在下用IL-6处理时组织蛋白酶B和L的产生，以及（2）其酶活性方面的作用。首先，我们通过瞬时转染Cav-1 siRNA建立了siRNA介导的体外下调Cav-1的系统。用IL-6/sIL-6R处理siRNA介导的Cav-1下调的细胞一段指定时间。然后，收集细胞裂解物，并检测IL-6诱导的信号通路、组织蛋白酶B和L的产生以及组织蛋白酶活性的测定。为了研究组织蛋白酶L的活性，在用组织蛋白酶B的特异性抑制剂CA-074Me预处理后测量组织蛋白酶-（B + L）的活性。我们发现IL-6/sIL-6R显著增强了HGFs中组织蛋白酶B和L的产生及活性。有趣的是，在用IL-6/sIL-6R处理的Cav-1下调的HGFs中，IL-6介导的p44/42 MAPK和JNK的磷酸化被显著抑制。此外，组织蛋白酶B和L的产生及活性也被显著抑制。重要的是，我们证明JNK抑制而非p44/42 MAPK抑制显著减少了IL-6/sIL-6R诱导的组织蛋白酶B和L的产生。综上所述，我们得出结论，IL-6/sIL-6R通过IL-6/sIL-6R介导的Cav-1-JNK-AP-1途径增强HGFs中组织蛋白酶B和L的产生。我们的研究结果表明，Cav-1可能是牙周炎中IL-6介导的组织降解的治疗靶点。
